Tazobactam arginine compositions

ABSTRACT

This disclosure provides compositions containing solid forms of tazobactam arginine, and methods of manufacturing and using these compositions.

TECHNICAL FIELD

This disclosure relates to solid forms of (2S,3S,5R)-3-((1H-1,2,3-triazol-1-yl)methyl)-3-methyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide (tazobactam), and related compositions and methods.

BACKGROUND

The crystal state of a compound may be important when the compound is used for pharmaceutical purposes. Compared with an amorphous solid, the solid physical properties of a crystalline compound may change, which may affect its suitability for pharmaceutical use. For example, a particular crystalline compound may overcome the disadvantage of other solid forms of the compound that readily absorb moisture (e.g., high hygroscopicity). For an ionic drug substance, high hygroscopicity may diminish the drug product's stability profile by a host of mechanisms, as the drug substance may have a propensity to absorb water. Water that is absorbed from the environment (packaging materials, exposure to air, or in the case of formulated products, from other materials), may lead to degradation products and/or impurities in a drug product or add to the cost of manufacturing the drug product with acceptably low levels of water.

One compound that can be obtained in amorphous or various crystalline salt forms is (2S,3S,5R)-3-((1H-1,2,3-triazol-1-yl)methyl)-3-methyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide, or tazobactam. There is a need for solid forms of tazobactam with reduced hygroscopicity for use in drug substance and drug product development.

SUMMARY

Solid forms of tazobactam (e.g., arginine salt forms of tazobactam) and compositions comprising these solid forms, are provided herein, in addition to various methods of preparing these compositions. Compared with previous crystalline forms of tazobactam, certain crystalline tazobactam arginine solid forms are provided herein that have the advantageous characteristic of being less hygroscopic. These crystalline tazobactam arginine solid forms can have good thermal stability and light stability in the process of preparation, packing, transportation and storage. Crystalline compounds of tazobactam arginine can also possess other properties that may be beneficial to the preparation of various drug formulations.

Crystalline tazobactam arginine, and hydrates and solvates thereof, can be obtained in various solid forms. Tazobactam arginine can be a salt consisting of the conjugate base of (2S,3S,5R)-3-((1H-1,2,3-triazol-1-yl)methyl)-3-methyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide (tazobactam) and the conjugate acid of (S)-2-amino-5-guanidinopentanoic acid (L-arginine) in a 1:1 ratio, as represented by the structure below.

In one aspect, provided herein is a particularly preferred crystalline tazobactam arginine solid form (designated herein as “polymorph Ia” or “tazobactam arginine polymorph Ia”), characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta at angles of 4.8°±0.2°, 8.9°±0.2°, 11.3°±0.2°, 14.9°±0.2°, 18.0°±0.2°, 19.4°±0.2°, 21.3°±0.2°, 22.8°±0.2°, and 24.3°±0.2°. The crystalline tazobactam arginine can be further characterized by a differential scanning calorimetry thermogram having a characteristic peak expressed in units of ° C. at a temperature in the range of about 209.2 to about 211.9. In another embodiment, the crystalline tazobactam arginine is further characterized by a thermogravimetry curve with an onset temperature of about 201.9° C.

Also provided are pharmaceutical compositions comprising a compound having a beta-lactam chemical sub-structure (e.g., a beta-lactam antibiotic compound) and crystalline tazobactam arginine (e.g., of the polymorph Ia solid form).

In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a crystalline tazobactam arginine compound (e.g., of the polymorph Ia solid form). The crystalline tazobactam arginine can be characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta at angles of 4.8°±0.2°, 8.9°±0.2°, 11.3°±0.2°, 14.9°±0.2°, 18.0°±0.2°, 19.4°±0.2°, 21.3°±0.2°, 22.8°±0.2°, and 24.3°±0.2°.

In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising an beta-lactam compound and a crystalline tazobactam arginine compound (e.g., of the polymorph Ia solid form). The crystalline tazobactam arginine can be characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta at angles of 4.8°±0.2°, 8.9°±0.2°, 11.3°±0.2°, 14.9°±0.2°, 18.0°±0.2°, 19.4°±0.2°, 21.3°±0.2°, 22.8°±0.2°, and 24.3°±0.2°.

In another aspect, provided herein is a method for detecting or identifying an agent that inhibits one or more beta-lactamase-producing organisms, said method comprising combining:

(a) a test agent;

(b) a composition comprising one or more beta-lactamase-producing organisms; and

(c) crystalline tazobactam arginine, wherein the crystalline tazobactam arginine is characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta at angles of 4.8°±0.2°, 8.9°±0.2°, 11.3°±0.2°, 14.9°±0.2°, 18.0°±0.2°, 19.4°±0.2°, 21.3°±0.2°, 22.8°±0.2° and 24.3°±0.2°; and detecting or measuring a change in the activity of the beta-lactamase-producing organisms, wherein a decrease in the activity of the beta-lactamase-producing organisms indicates that the test agent inhibits the beta-lactamase-producing organisms.

As used herein, the phrase “degrees 2-Theta±0.2°” indicates that each subsequently listed angle has an error of ±0.2°; and the phrase “degrees 2-Theta±0.1°” indicates that each subsequently listed angle has an error of ±0.1°. For example, the phrase “degrees 2-Theta±0.2° at angles of 1, 2 and 3” is equivalent to the phrase “degrees 2-Theta at angles of 1±0.2°, 2±0.2° and 3±0.2°”.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the X-ray powder diffraction pattern of polymorph Ia (Example 2).

FIG. 2 depicts the differential scanning calorimetry (DSC) thermogram of polymorph Ia.

FIG. 3 depicts the thermogravimetry curve of polymorph Ia.

FIG. 4 depicts the X-ray powder diffraction pattern of polymorph Ib.

FIG. 5 depicts the X-ray powder diffraction pattern of tazobactam arginine amorphous.

FIG. 6 depicts the X-ray power diffraction pattern of polymorph Ia (Example 4).

FIG. 7 depicts the ¹H-NMR spectrum of polymorph Ia.

FIG. 8 depicts the DVS (dynamic vapor sorption) graph of tazobactam arginine amorphous (Example 6a).

FIG. 9 depicts the DVS graph of polymorph Ia (Example 6b).

FIG. 10 depicts the DVS graph of amorphous tazobactam sodium (Example 6c).

DETAILED DESCRIPTION

Tazobactam arginine may occur in an amorphous solid form or in a crystalline solid form. Crystalline solid forms of tazobactam arginine may exist in one or more unique polymorph forms, which may additionally comprise one or more equivalents of water or solvent (i.e., hydrates or solvates, respectively).

Tazobactam arginine is the salt of the conjugate base of tazobactam and the conjugate acid of (S)-2-amino-5-guanidinopentanoic acid (L-arginine) in a 1:1 ratio, as represented by the structure below.

Accordingly, provided herein is crystalline tazobactam arginine, and hydrates and solvates thereof. In particular, provided herein is crystalline tazobactam arginine polymorph Ia, also referred to herein as “polymorph Ia” or “tazobactam arginine polymorph Ia”, and crystalline tazobactam arginine polymorph Ib, also referred to herein as “polymorph Ib” or “tazobactam arginine polymorph Ib”.

Polymorphism

The ability of a substance to exist in more than one crystal form is defined as polymorphism; the different crystal forms of a particular substance are referred to as “polymorphs.” In general, polymorphism is affected by the ability of a molecule of a substance to change its conformation or to form different intermolecular or intra-molecular interactions, particularly hydrogen bonds, which is reflected in different atom arrangements in the crystal lattices of different polymorphs. In contrast, the overall external form of a substance is known as “morphology,” which refers to the external shape of the crystal and the planes present, without reference to the internal structure. Crystals can display different morphology based on different conditions, such as, for example, growth rate, stirring, and the presence of impurities.

The different polymorphs of a substance can possess different energies of the crystal lattice and, thus, in solid state they can show different physical properties such as form, density, melting point, color, stability, solubility, dissolution rate, etc., which can, in turn, affect the stability, dissolution rate and/or bioavailability of a given polymorph and its suitability for use as a pharmaceutical and in pharmaceutical compositions.

Access to different polymorphs of tazobactam arginine is desirable for other reasons as well. One such reason is that different polymorphs of a compound (e.g., tazobactam arginine) can incorporate different impurities, or chemical residues, upon crystallization. Certain polymorphs incorporate very little, or no, chemical residues. Accordingly, the formation of certain polymorph forms of a compound may result in purification of the compound.

Tazobactam arginine polymorph Ia exhibits low hygroscopicity relative to amorphous tazobactam arginine and amorphous tazobactam sodium. Low hygroscopicity of a solid compound is desirable for several reasons. For example, compounds that are highly hygroscopic may be chemically unstable, or unsuitable for formulating as a drug product due to changes of the drug form's physical characteristics (e.g., bulk density, dissolution rate, etc.) that can occur if it is stored in settings with varying relative humidity. Also, hygroscopicity can impact large-scale manufacturing and handling of a compound. For example, it may be difficult to determine the true weight of a hygroscopic active agent when preparing a pharmaceutical composition comprising that agent.

Characterization of Polymorphs

In certain embodiments, the compounds of the invention are identifiable on the basis of characteristic peaks in an X-ray powder diffraction analysis. X-ray powder diffraction, also referred to as XRPD, is a scientific technique using X-ray, neutron, or electron diffraction on powder, microcrystalline, or other solid materials for structural characterization of the materials.

One embodiment of crystalline tazobactam arginine is referred to as polymorph Ia (also referred to herein as “tazobactam arginine polymorph Ia”) and is characterized by an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-Theta at angles selected from 8.9°±0.2°, 18.0°±0.2° and 21.3°±0.2°. In another embodiment, polymorph Ia is characterized by an X-ray powder diffraction pattern having one or more peaks expressed in degrees 2-Theta at angles selected from 4.8°±0.2°, 11.3°±0.2° and 14.9°±0.2°. In still another embodiment, polymorph Ia is characterized by an X-ray powder diffraction pattern having one or more peaks expressed in degrees 2-Theta at angles selected from 19.4°±0.2°, 22.8°±0.2° and 24.3°±0.2°.

In another embodiment, polymorph Ia is characterized by an X-ray powder diffraction pattern having 3-6 peaks expressed in degrees 2-Theta at angles selected from 8.9°±0.2°, 18.0°±0.2°, 21.3°±0.2°, 4.8°±0.2°, 11.3°±0.2°, 14.9°±0.2°, 19.4°±0.2°, 22.8°±0.2° and 24.3°±0.2°. In a particular embodiment, polymorph Ia is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-Theta at angles of 8.9°±0.2°, 18.0°±0.2° and 21.3°±0.2°.

In still another embodiment, polymorph Ia is characterized by an X-ray powder diffraction pattern having 6-9 peaks expressed in degrees 2-Theta at angles selected from 8.9°±0.2°, 18.0°±0.2°, 21.3°±0.2°, 4.8°±0.2°, 11.3°±0.2°, 14.9°±0.2°, 19.4°±0.2°, 22.8°±0.2° and 24.3°±0.2°. In a particular embodiment, polymorph Ia is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-Theta at angles of 4.8°±0.2°, 8.9°±0.2°, 11.3°±0.2°, 14.9°±0.2°, 18.0°±0.2°, 19.4°±0.2°, 21.3°±0.2° 22.8°±0.2° and 24.3°±0.2°.

In still another embodiment, provided herein is a composition comprising crystalline tazobactam arginine characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta±0.2° at angles of 4.8°, 8.9°, 11.3°, 14.9°, 18.0°, 19.4°, 21.3°, 22.8° and 24.3°.

In still another embodiment, provided herein is a composition comprising crystalline tazobactam arginine characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta±0.1° at angles of 4.8°, 8.9°, 11.3°, 14.9°, 18.0°, 19.4°, 21.3°, 22.8° and 24.3°.

In still another embodiment, provided herein is a composition comprising crystalline tazobactam arginine characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta at angles of about 4.8°, 8.9°, 11.3°, 14.9°, 18.0°, 19.4°, 21.3°, 22.8° and 24.3°.

In one embodiment, polymorph Ia is characterized by an X-ray powder diffraction pattern having peaks substantially in accordance with FIG. 1. In another embodiment, polymorph Ia is characterized by an X-ray powder diffraction pattern having peaks substantially in accordance with Table 1.

The compounds of the invention may also be defined by their differential scanning calorimetry (DSC) thermograms. In one embodiment, polymorph Ia is characterized by a differential scanning calorimetry thermogram having a characteristic peak expressed in units of ° C. at a temperature of 209.2±3. In another embodiment, polymorph Ia is characterized by a differential scanning calorimetry thermogram having a characteristic peak expressed in units of ° C. in the range of about 209.2 to about 211.9. In a particular embodiment, polymorph Ia is characterized by a differential scanning calorimetry thermogram substantially in accordance with FIG. 2.

The compounds of the invention can be also be defined by their thermogravimetry (TG) signals. In one embodiment, polymorph Ia is characterized by a thermogravimetry curve with an onset temperature of 201.9° C.±3° C. In another embodiment, polymorph Ia is characterized by a thermogravimetry curve with an onset temperature of about 201.9° C. In a particular embodiment, polymorph Ia is characterized by a thermogravimetry curve substantially in accordance with FIG. 3.

In certain embodiments, polymorph Ia may contain impurities. Non-limiting examples of impurities include undesired polymorph forms, or residual organic and inorganic molecules such as solvents, water or salts.

In another embodiment, polymorph Ia is substantially free from impurities. In another embodiment, polymorph Ia contains less than 10% by weight total impurities. In another embodiment, polymorph Ia contains less than 5% by weight total impurities. In another embodiment, polymorph Ia contains less than 1% by weight total impurities. In yet another embodiment, polymorph Ia contains less than 0.1% by weight total impurities.

In another aspect, provided herein is crystalline tazobactam arginine polymorph Ib. In one embodiment, polymorph Ib is tazobactam arginine trihydrate. In another embodiment, crystalline tazobactam polymorph Ib is characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta at angles of 4.4°±0.2°, 9.7°±0.2°, 17.3°±0.2°, 20.2°±0.2°, and 22.0°±0.2°. In a particular embodiment, polymorph Ib is characterized by an X-ray powder diffraction pattern having peaks substantially in accordance with FIG. 4.

In another aspect, provided herein is a composition comprising one or more compounds selected from amorphous tazobactam arginine, polymorph Ia and polymorph Ib. In one embodiment, the composition comprises one or more compounds selected from tazobactam arginine and polymorph Ia.

In certain embodiments, polymorph Ia is a crystalline solid substantially free of amorphous tazobactam arginine. As used herein, the term “substantially free of amorphous tazobactam arginine” means that the compound contains no significant amount of amorphous tazobactam arginine. In certain embodiments, at least about 95% by weight of crystalline polymorph Ia is present. In still other embodiments of the invention, at least about 99% by weight of crystalline polymorph Ia is present.

In another embodiment, polymorph Ia is substantially free from polymorph Ib. As used herein, the term “substantially free of polymorph Ib” means that the compound contains no significant amount of polymorph Ib. In certain embodiments, at least about 95% by weight of crystalline polymorph Ia is present. In still other embodiments of the invention, at least about 99% by weight of crystalline polymorph Ia is present.

Processes and Methods

Provided herein is a method of making crystalline tazobactam arginine comprising:

(1) combining tazobactam acid, arginine and a solvent, such that a solution of tazobactam arginine is formed; and

(2) combining an antisolvent with the solution, wherein the antisolvent is miscible with the solvent and wherein tazobactam arginine is partially or completely insoluble in the antisolvent, such that crystalline tazobactam arginine precipitates from the solution.

In step (1), tazobactam acid may first be combined with the solvent, and the resulting mixture then combined with arginine. Alternatively, arginine may first be combined with the solvent, and the resulting mixture then combined with tazobactam acid. Alternatively, tazobactam acid and arginine may be combined, and the resulting mixture then combined with the solvent.

In one embodiment, the above method is a method of making tazobactam arginine polymorph Ia, said method further comprising: (3) drying the precipitated crystalline tazobactam arginine to afford polymorph Ia.

In one embodiment, the antisolvent is added to the solution of tazobactam arginine. In another embodiment, the solution of tazobactam arginine is added to the antisolvent. In certain embodiments, the solvent is selected from the group consisting of water and acetic acid. In certain embodiments, the antisolvent is selected from the group consisting of alcohols, ethers, esters, ketones, nitriles, amides, nitroalkanes, nitroarenes, substituted or unsubstituted aromatic solvents, substituted or unsubstituted aliphatic solvents and mixtures thereof. In certain embodiments, the antisolvent is selected from the group consisting of acetone, acetonitrile, 1-butanol, cyclohexane, dichloromethane, diisopropyl ether, dimethylformamide, dimethylsulfoxide, 1,4-dioxane, ethanol, ethyl acetate, heptanes, methanol, isopropyl acetate, methyl ethyl ketone, methyl isobutyl ketone, N-methyl-2-pyrrolidinone, nitromethane, 2-propanol, tert-butylmethyl ether, tetrahydrofuran, toluene and mixtures thereof. Experiments determining solubility of crystalline tazobactam arginine in a variety of solvents are described in Experiment 6, and results are summarized in Table 3. In a preferred embodiment, the solvent is water. In another preferred embodiment, the antisolvent is acetone. In yet another preferred embodiment, the antisolvent is isopropanol.

Accordingly, provided herein is a method of making crystalline tazobactam arginine comprising:

(1) combining tazobactam acid, arginine and water, such that an aqueous solution of tazobactam arginine is formed; and

(2) combining acetone with the aqueous solution, such that crystalline tazobactam arginine precipitates from the solution.

In one embodiment, the above method is a method of making tazobactam arginine polymorph Ia, said method further comprising: (3) drying the precipitated crystalline tazobactam arginine to afford polymorph Ia.

In another aspect, provided herein is a method of making crystalline tazobactam arginine (e.g., polymorph Ia) comprising:

(1) combining tazobactam arginine and a solvent, such that a solution of tazobactam arginine is formed; and

(2) combining an antisolvent with the solution, wherein the antisolvent is miscible with the solvent and wherein tazobactam arginine is partially or completely insoluble in the antisolvent, such that crystalline tazobactam arginine precipitates from the solution.

In another aspect, provided herein is a method of making crystalline tazobactam arginine comprising:

(1) combining tazobactam acid, arginine and a solvent/antisolvent mixture, such that a solution of tazobactam arginine is formed; and

(2) combining an antisolvent with the solution, wherein the antisolvent is miscible with the solvent and wherein tazobactam arginine is partially or completely insoluble in the antisolvent, such that crystalline tazobactam arginine precipitates from the solution.

In certain embodiments, any one of the above methods is a method of making tazobactam arginine polymorph Ia and the method further comprises: (3) drying the precipitated crystalline tazobactam arginine to afford polymorph Ia.

In another aspect, provided herein is a method of making crystalline tazobactam arginine comprising:

(1) combining a compound according to formula (I), a compound according to formula (II), and a solvent, such that a solution comprising tazobactam arginine is formed, and such that crystalline tazobactam arginine precipitates from the solution.

In one embodiment, Y is a metal atom or ion and X— is a halide ion.

In still another aspect, provided herein is a method of making crystalline tazobactam arginine comprising:

(1) combining a compound according for formula (I), a compound according to formula (II), and a solvent, such that a solution comprising tazobactam arginine is formed; and

(2) combining an antisolvent with the solution, wherein the antisolvent is miscible with the solvent and wherein tazobactam arginine is partially or completely insoluble in the antisolvent, such that crystalline tazobactam arginine precipitates from the solution. In one embodiment, Y is a metal atom or ion and X— is a halogen ion.

In another aspect, provided herein is crystalline tazobactam arginine produced according to the any one of the preceding methods. In another aspect, provided herein is crystalline tazobactam arginine obtainable by any one of the preceding methods.

The processes and methods described herein may also further comprise adding one or more seed crystals of crystalline tazobactam arginine (e.g., polymorph Ia or polymorph Ib).

As used herein, the verb “precipitate” refers to the formation of a solid substance from a solution containing the same substance. A substance which precipitates from solution may be amorphous or crystalline. Precipitation may occur under a variety of conditions known to those of skill in the art, including the treatment of a solution of a solute (e.g., solute A in solvent B) with an antisolvent (i.e., a solvent that is miscible with solvent B, but does not dissolve solute A). Non-limiting examples of solvent/antisolvent pairs include water/acetone and water/isopropanol.

Pharmaceutical Compositions

In one aspect, provided herein is a pharmaceutical composition comprising tazobactam arginine polymorph Ia.

In another aspect, provided herein is a pharmaceutical composition comprising crystalline tazobactam arginine, hydrates or solvates thereof, and one or more beta-lactam compounds, and a pharmaceutically acceptable carrier or diluent. In one embodiment, the pharmaceutical composition is useful for treating a bacterial infection. The bacterial infection can result from either gram-negative or gram-positive organisms. In one embodiment, the crystalline tazobactam arginine is polymorph Ia. Polymorph Ia is characterized as described above.

In yet another aspect, provided herein is a pharmaceutical composition prepared by a method comprising the step of combining crystalline tazobactam arginine, or hydrates or solvates thereof, and a beta-lactam compound. In one embodiment, the crystalline tazobactam arginine is polymorph Ia. Polymorph Ia is characterized as described above.

A “beta-lactam compound” is a compound possessing one or more beta-lactam moieties, i.e.,

substituted one or more times as valency permits. In certain non-limiting embodiments the beta-lactam compounds described herein can be selected from the group consisting of penicillins, cephalosporins, carbapenems, and combinations thereof. In certain embodiments, said one or more beta-lactam compounds are selected from the compounds listed in Table 2, and pharmaceutically acceptable isomers, salts, esters, hydrates, solvates, or combinations thereof.

The following compounds are listed in Table 2:

-   (2S,5R,6R)-6-[(R)-2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido)-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic     acid; -   (2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-zabicyclo[3.2.0]heptane-2-carboxylic     acid; -   (5R,6S)-6-[(1R)-1-hydroxyethyl]-3-({2-[(iminomethyl)amino]ethyl}thio)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic     acid; -   (5R,6S)-6-((R)-1-hydroxyethyl)-7-oxo-3-((R)-tetrahydrofuran-2-yl)-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic     acid; -   (2S,5R,6R)-6-{[3-(2-chlorophenyl)-5-methyl-oxazole-4-carbonyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic     acid; -   (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-carboxypropan-2-yloxyimino)acetamido)-8-oxo-3-(pyridinium-1-ylmethyl)-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate; -   6R,7R,Z)-3-(acetoxymethyl)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic     acid; -   (6R,7R)-7-[(2Z)-2-ethoxyimino-2-[5-(phosphonoamino)-1,2,4-thiadiazol-3-yl]acetyl]amino]-3-[4-(1-methylpyridin-1-ium-4-yl)-1,3-thiazol-2-yl]sulfanyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate; -   (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((1-methylpyrrolidinium-1-yl)methyl)-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate; -   (6R,7R)-3-{[(aminocarbonyl)oxy]methyl}-7-{[(2Z)-2-(2-furyl)-2-(methoxyimino)     acetyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic     acid; -   (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino}-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)thio]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic     acid; -   (2S,5R,6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic     acid; -   3-[5-(dimethylcarbamoyl)pyrrolidin-2-yl]sulfanyl-6-(1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic     acid.

The skilled practitioner will recognize that the beta-lactam compounds described herein have one or more acidic moieties (e.g., carboxylic acid moieties) and/or one or more basic moieties (e.g., amine moieties). Said moieties may be protonated or deprotonated as a function of pKa or pKb of the moiety and the pH of the compound's environment. All salt forms resulting from the protonation or deprotonation of a beta-lactam compound are contemplated by the instant disclosure.

In a particular embodiment, the pharmaceutical composition comprises polymorph Ia and (2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-zabicyclo[3.2.0]heptane-2-carboxylic acid or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (5R,6S)-6-[(1R)-1-hydroxyethyl]-3-({2-[(iminomethyl)amino]ethyl}thio)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (5R,6S)-6-((R)-1-hydroxyethyl)-7-oxo-3-((R)-tetrahydrofuran-2-yl)-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (2S,5R,6R)-6-{[3-(2-chlorophenyl)-5-methyl-oxazole-4-carbonyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-carboxypropan-2-yloxyimino)acetamido)-8-oxo-3-(pyridinium-1-ylmethyl)-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (6R,7R,Z)-3-(acetoxymethyl)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (6R,7R)-7-[(2Z)-2-ethoxyimino-2-[5-(phosphonoamino)-1,2,4-thiadiazol-3-yl]acetyl]amino]-3-[4-(1-methylpyridin-1-ium-4-yl)-1,3-thiazol-2-yl]sulfanyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((1-methylpyrrolidinium-1-yl)methyl)-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (6R,7R)-3-{[(aminocarbonyl)oxy]methyl}-7-{[(2Z)-2-(2-furyl)-2-(methoxyimino)acetyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino}-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)thio]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (2S,5R,6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and (2S,5R,6R)-6-[(R)-2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido)-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

In another particular embodiment, the pharmaceutical composition comprises polymorph Ia and 3-[5-(dimethylcarbamoyl)pyrrolidin-2-yl]sulfanyl-6-(1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof, and a pharmaceutically acceptable carrier or diluent.

Pharmaceutical compositions comprising compounds of the invention can be identified by comparison of the compositions' X-ray powder diffraction patterns to an X-ray powder diffraction pattern of a compound of the invention. It will be appreciated that pharmaceutical compositions comprising a compound of the invention may exhibit non-identical X-ray powder diffraction patterns as compared to an X-ray powder diffraction pattern of a pure compound of the invention.

The term “pharmaceutical composition” includes preparations suitable for administration to mammals, e.g., humans. When the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1% to 99.9% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

The pharmaceutical compositions described herein can be formulated to have any concentration desired (i.e., any concentration of crystalline tazobactam arginine, or a hydrate or solvate thereof, and any concentration of a beta-lactam compound). In some embodiments, the composition is formulated such that it comprises at least a therapeutically effective amount of both compounds (i.e., a therapeutically effective amount of the combination of crystalline tazobactam arginine, or a hydrate or solvate thereof, and the beta-lactam compound). In some embodiments, the composition is formulated such that it would not cause one or more unwanted side effects.

The compounds of the invention (i.e., polymorphs, hydrates and solvates of tazobactam arginine) can be combined with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques. As used herein, “pharmaceutically acceptable carrier” may include any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Eighteenth Edition, A. R. Gennaro (Mack Publishing Co., Easton, Pa., 1990) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline (“normal saline”); Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, trehalose, or reducing or non-reducing sugars, 5% dextrose (D5W), preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

Furthermore, the carrier may take a wide variety of forms depending on the form of the preparation desired for administration, e.g. oral, nasal, rectal, vaginal, parenteral (including intravenous injections or infusions). In preparing compositions for oral dosage form any of the usual pharmaceutical media may be employed. Usual pharmaceutical media include, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as for example, suspensions, solutions, emulsions and elixirs); aerosols; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like, in the case of oral solid preparations (such as for example, powders, capsules, and tablets).

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Pharmaceutical compositions include those suitable for oral, sublingual, nasal rectal, vaginal, topical, buccal and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route will depend on the nature and severity of the condition being treated. The compositions may be conveniently presented in unit dosage form, and prepared by any of the methods well known in the art of pharmacy. In certain embodiments, the pharmaceutical composition is formulated for oral administration in the form of a pill, capsule, lozenge or tablet. In other embodiments, the pharmaceutical composition is in the form of a suspension.

The pharmaceutical compositions disclosed herein can be prepared via lyophilization. As is known to those skilled in the art, lyophilization is a process of drying in which water is sublimed from a frozen solution of one or more solutes. Specific methods of lyophilization are described in Remington's Pharmaceutical Sciences, Chapter 84, page 1565, Eighteenth Edition, A. R. Gennaro, (Mack Publishing Co., Easton, Pa., 1990).

In a preferred embodiment, pharmaceutical compositions comprising crystalline tazobactam arginine (e.g., polymorph Ia) and one or more beta-lactam compounds are formulated for parenteral administration. In another preferred embodiment, pharmaceutical compositions comprising tazobactam arginine and one or more beta-lactam compounds are formulated for oral administration.

Methods of Treatment

Tazobactam arginine inhibits or decreases the activity of beta-lactamases (e.g., bacterial beta-lactamases), and can be combined with beta-lactam compounds (e.g., antibiotics), thereby broadening the spectrum of the beta-lactam compound and increasing the beta-lactam compound's efficacy against organisms that produce beta-lactamase. A compound or a composition possesses efficacy against an organism if it kills or weakens the organism, or inhibits or prevents reproduction the organism.

In one aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of tazobactam arginine polymorph Ia.

In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising tazobactam arginine polymorph Ia.

In yet another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising crystalline tazobactam arginine and one or more beta-lactam compounds, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof. In one embodiment, the mammal is human. In another embodiment, tazobactam arginine is polymorph Ia. In yet another embodiment, said one or more beta-lactam compounds are selected from the group consisting of penicillins, cephalosporins, carbapenems, and combinations thereof. In certain embodiments, the beta-lactam compound is selected from the compounds listed in Table 2, and pharmaceutically acceptable isomers, salts, esters, hydrates, solvates, or combinations thereof.

The following compounds are listed in Table 2: (2S,5R,6R)-6-[(R)-2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido)-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid; (2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-zabicyclo[3.2.0]heptane-2-carboxylic acid; (5R,6S)-6-[(1R)-1-hydroxyethyl]-3-({2-[(iminomethyl)amino]ethyl}thio)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid; (5R,6S)-6-((R)-1-hydroxyethyl)-7-oxo-3-((R)-tetrahydrofuran-2-yl)-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid; (2S,5R,6R)-6-{[3-(2-chlorophenyl)-5-methyl-oxazole-4-carbonyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid; (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-carboxypropan-2-yloxyimino)acetamido)-8-oxo-3-(pyridinium-1-ylmethyl)-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate; (6R,7R,Z)-3-(acetoxymethyl)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid; (6R,7R)-7-[(2Z)-2-ethoxyimino-2-[5-(phosphonoamino)-1,2,4-thiadiazol-3-yl]acetyl]amino]-3-[4-(1-methylpyridin-1-ium-4-yl)-1,3-thiazol-2-yl]sulfanyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate; (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((1-methylpyrrolidinium-1-yl)methyl)-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate; (6R,7R)-3-{[(aminocarbonyl)oxy]methyl}-7-{[(2Z)-2-(2-furyl)-2-(methoxyimino) acetyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid; (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino}-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)thio]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid; (2S,5R,6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid; 3-[5-(dimethylcarbamoyl)pyrrolidin-2-yl]sulfanyl-6-(1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.

In a particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-zabicyclo[3.2.0]heptane-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (5R,6S)-6-[(1R)-1-hydroxyethyl]-3-({2-[(iminomethyl)amino]ethyl}thio)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (5R,6S)-6-((R)-1-hydroxyethyl)-7-oxo-3-((R)-tetrahydrofuran-2-yl)-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (2S,5R,6R)-6-{[3-(2-chlorophenyl)-5-methyl-oxazole-4-carbonyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-carboxypropan-2-yloxyimino)acetamido)-8-oxo-3-(pyridinium-1-ylmethyl)-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (6R,7R,Z)-3-(acetoxymethyl)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (6R,7R)-7-[(2Z)-2-ethoxyimino-2-[5-(phosphonoamino)-1,2,4-thiadiazol-3-yl]acetyl]amino]-3-[4-(1-methylpyridin-1-ium-4-yl)-1,3-thiazol-2-yl]sulfanyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((1-methylpyrrolidinium-1-yl)methyl)-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (6R,7R)-3-{[(aminocarbonyl)oxy]methyl}-7-{[(2Z)-2-(2-furyl)-2-(methoxyimino) acetyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino}-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)thio]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (2S,5R,6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and 3-[5-(dimethylcarbamoyl)pyrrolidin-2-yl]sulfanyl-6-(1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another particular embodiment of the method, the pharmaceutical composition comprises polymorph Ia and (2S,5R,6R)-6-[(R)-2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido)-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof. In another particular embodiment, the bacterial infection is caused by bacteria that are susceptible to the composition comprising polymorph Ia and (2S,5R,6R)-6-[(R)-2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido)-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, or a pharmaceutically acceptable isomer, salt, ester, hydrate, solvate, or combination thereof.

In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising an antibiotic and a crystalline tazobactam arginine compound (e.g., of the polymorph Ia solid form). The crystalline tazobactam arginine can be characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta at angles of 4.8°±0.2°, 8.9°±0.2°, 11.3°±0.2°, 14.9°±0.2°, 18.0°±0.2°, 19.4°±0.2°, 21.3°±0.2°, 22.8°±0.2°, and 24.3°±0.2°.

Non-limiting examples of bacterial infections that can be treated by the methods of the invention include infections caused by: aerobic and facultative gram-positive microorganisms (e.g., Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Viridans group streptococci), aerobic and facultative gram-negative microorganisms (e.g., Acinetobacter baumanii, Escherichia coli, Haemophilus influenza, Klebsiella pneumonia, Pseudomonas aeruginosa, Citrobacter koseri, Moraxella catarrhalis, Morganella morganii, Neisseria gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Serratia marcescens, Providencia stuartii, Providencia rettgeri, Salmonella enterica), gram-positive anaerobes (Clostridium perfringens), and gram-negative anaerobes (e.g., Bacteroides fragilis group (e.g., B. fragilis, B. ovatus, B. thetaiotaomicron, and B. vulgates), Bacteroides distasonis, Prevotella melaminogenica).

In certain embodiments of the methods described herein, bacterial infection resulting from beta-lactamase-producing organisms are treated or controlled. Non-limiting examples of beta-lactamase-producing organisms include:

(1) ESBL (extended-spectrum beta-lactamase)-producing organisms selected from the group consisting of Enterobacteriaceae spp.: Escherichia coli, Klebsiella spp. (including K. pneumoniae and K. oxytoca), Proteus mirabilis, Proteus vulgaris, Enterobacter spp., Serratia spp., Citrobacter spp., Pseudomonas spp., Acinetobacter spp.) and Bacteroides spp.;

(2) CSBL (conventional-spectrum beta-lactamase)-producing organisms, known to those of skill in the art; and

(3) Inducible-AmpC-type beta-lactamases, such as Citrobacter spp., Serratia spp., Morganella morganii, Proteus vulgaris, and Enterobacter cloacae.

In certain embodiments of the methods described herein, bacterial infection is associated with one or more of the following conditions:

Appendicitis (complicated by rupture or abscess) and peritonitis caused by piperacillin-resistant beta-lactamase producing strains of Escherichia coli or the following members of the Bacteroides fragilis group: B. fragilis, B. ovatus, B. thetaiotaomicron, or B. vulgates;

Uncomplicated and complicated skin and skin structure infections, including cellulitis, cutaneous abscesses, and ischemic/diabetic foot infections caused by piperacillin-resistant, beta-lactamase producing strains of Staphylococcus aureus;

Postpartum endometritis or pelvic inflammatory disease caused by piperacillin-resistant, beta-lactamase producing strains of Escherichia coli;

Community-acquired pneumonia (moderate severity only) caused by piperacillin-resistant, beta-lactamase producing strains of Haemophilus influenza;

Nosocomial pneumonia (moderate to severe) caused by piperacillin-resistant, beta-lactamase producing strains of Staphylococcus aureus and by Acinetobacter baumanii, Haemophilus influenzae, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

Complicated intra-abdominal infections; Complicated urinary tract infections (cUTIs); Acute Pyelonephritis; Systemic Inflammatory Response Syndrome (SIRS).

Also provided herein is the use of a crystalline tazobactam arginine, and hydrates and solvates thereof, in combination with one or more beta-lactam compounds, for the manufacture of a medicament for the treatment of bacterial infection. The bacterial infection can result from either gram-negative or gram-positive organisms. In one embodiment, the crystalline tazobactam arginine is polymorph Ia. Polymorph Ia is characterized as described above. Said one or more beta-lactam compounds can be selected from the group consisting of penicillins, cephalosporins, carbapenems, and combinations thereof. In certain embodiments, said one or more beta-lactam compounds are selected from the compounds listed in Table 2, and pharmaceutically acceptable isomers, salts, esters, hydrates, solvates, or combinations thereof.

Also provided herein is tazobactam arginine, and hydrates and solvates thereof, for use in a method of treating bacterial infection. In some embodiments, a combination of tazobactam arginine and one or more beta-lactam compounds is used in said method.

As used herein, “treating”, “treat” or “treatment” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a pharmaceutical composition of the present invention to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.

By a “therapeutically effective amount” of a compound of the invention is meant a sufficient amount of the compound to treat the disorder (e.g., bacterial infection). The specific therapeutically effective amount that is required for the treatment of any particular patient or organism (e.g., a mammal) will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound or composition employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see, for example, Goodman and Gilman's, “The Pharmacological Basis of Therapeutics”, Tenth Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001, which is incorporated herein by reference in its entirety). The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.

Assays

Provided herein is a method for detecting or identifying an agent that will inhibit one or more beta-lactamase-producing organisms, said method comprising combining:

(a) a test agent;

(b) a composition comprising one or more beta-lactamase-producing organisms; and

(c) a beta-lactamase inhibitor; and

detecting or measuring a change in the activity of the beta-lactamase-producing organisms, wherein a decrease in the acitivity of the beta-lactamase-producing organisms indicates that the test agent inhibits the beta-lactamase-producing organisms.

As used in the above method, “activity” refers to the ability of the beta-lactamase-producing organism to reproduce and/or infect another organism, or “activity” refers to the presence of an indicator of the ability of the beta-lactamase-producing organism to reproduce and/or infect another organism. Methods for detecting and/or measuring changes in the activity of beta-lactamase-producing organisms are known to those of skill in the art.

In another aspect, provided herein is a method of determining the susceptibility of a beta-lactamase-producing organism to a composition comprising a beta-lactam compound and a beta-lactamase inhibitor. The in vitro activity of compositions of the subject invention may be assessed by standard testing procedures. Non-limiting examples of such a procedure include the Kirby-Bauer method, the Stokes test, the E-test, broth dilution and agar dilution for determination of minimum inhibitory concentration (MIC), as described in “Approved Standard. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically,” 3.sup.rd ed., published 1993 by the National Committee for Clinical Laboratory standards, Villanova, Pa., USA. In certain embodiments, the methods described herein are performed using automation (e.g., Siemens' MicroScan Systems).

In one embodiment of the above methods, the beta-lactamase inhibitor is tazobactam arginine. In a preferred embodiment, the beta-lactamase inhibitor is tazobactam arginine polymorph Ia.

The test agent can be selected from the group consisting of penicillins, cephalosporins, carbapenems, and combinations thereof. In some embodiments, the test agent is selected from the compounds listed in Table 2, and pharmaceutically acceptable isomers, salts, esters, hydrates, solvates, or combinations thereof.

In certain embodiments of the methods described herein, beta-lactamase-producing organisms are selected from the group comprising:

(1) ESBL (extended-spectrum beta-lactamase)-producing organisms selected from the group consisting of Enterobacteriaceae spp.: Escherichia coli, Klebsiella spp. (including K. pneumoniae and K. oxytoca), Proteus mirabilis, Proteus vulgaris, Enterobacter spp., Serratia spp., Citrobacter spp.) and Bacteroides spp.;

(2) CSBL (conventional-spectrum beta-lactamase)-producing organisms, known to those of skill in the art; and

(3) Inducible-AmpC-type beta-lactamases, such as Citrobacter spp., Serratia spp., Morganella morganii, Proteus vulgaris, and Enterobacter cloacae.

EXAMPLES Example 1 Preparation of Tazobactam Arginine Amorphous

L-arginine (2.9 g) was dissolved in 100 mL of deionized water and 5.0 g of tazobactam acid was then added, resulting a homogenous solution. The solution was lyophilized overnight to yield tazobactam arginine amorphous as an off-white solid (8.0 g). The XRPD spectrum of the tazobactam arginine amorphous is depicted in FIG. 5.

Example 2 Preparation of Tazobactam Arginine Crystalline Polymorph Ia

Tazobactam arginine amorphous (1.00 g) was dissolved in 10.0 mL of deionized water. 30 mL of acetone was added to the aqueous solution by drop-wise addition. The mixture was allowed to sit overnight at ambient temperature, resulting in white fine needles. After filtration and vacuum drying for 4 hours, tazobactam arginine polymorph Ia (516 mg) was obtained. The XRPD spectrum of the tazobactam arginine polymorph Ia is depicted in FIG. 1.

Example 3 Preparation of Tazobactam Arginine Crystalline Polymorph Ia

L-arginine (1.16 g) was charged to the reactor and dissolved in a mixture of deionized water-acetone (1:1, v/v, 40 mL) by mechanically stirring at 300 rpm, 30° C. over 5 min. Tazobactam acid (2.00 g) was then added to the above solution over 2 min with the observed pH change from 10.5±0.5 to 5.9±0.5. After adding 34.5 mL of acetone over 30 min at 30° C., crystallization started, then the suspension was cooled down to 15° C. at 0.1° C./min and kept stirring at 15° C. for additional 1.5 hr. White fine needle crystals were filtered out and vacuum dried for 2 hours at 35° C. to obtain 2.59 g (82% yield) of tazobactam arginine polymorph Ia.

Example 4 Preparation of Tazobactam Arginine Crystalline Polymorph Ia—20 g Scale

1) 20 g of tazobactam acid and 1 equivalent of L-arginine was placed into a jacketed, glass, 1 liter laboratory reactor with an overhead stirrer.

2) 5 volumes (relative to tazobactam acid weight) of ambient temperature (ca. 22° C.) water was added to the reactor and stirred at 480 rpm. The reactor temperature was maintained between 23-27° C. with a target temperature of 25° C. The reaction was stirred for 10-15 minutes in order to obtain complete dissolution. Stirring at 480 rpm was maintained throughout the reaction.

3) 0.75 vol. ambient temperature deionised water was added to the reactor. This step was included to account for the sterile filtration and washing step required in the process.

4) 8 volumes (160 ml) of acetone was added over 10 minutes.

5) The reaction mixture was aged for 15 minutes, at which point precipitation of tazobactam arginine begins.

6) A controlled cool was started from 25° C. down to 10° C. over 1.5 hours at a rate of 10° C./hour.

7) After reaching 10° C., 18 volumes of acetone was added over two hours (i.e., total acetone added throughout reaction was 26 volumes).

8) After the acetone addition, the reaction mixture was allowed to age for 1 hour.

9) The reaction mixture was filtered and washed with 5 volumes of cold acetone (ca. 4° C.).

10) The resulting solid was dried at ambient (ca. 22° C.) under vacuum and nitrogen bleed for 24 hours with regular mixing of the solid, to furnish polymorph Ia in 90% yield. The XRPD spectrum of the tazobactam arginine polymorph Ia is depicted in FIG. 6.

The ¹H-NMR spectrum (FIG. 7) of polymorph Ia indicates a stoichiometry of 1:1 tazobactam acid:arginine.

Example 5 Preparation of Tazobactam Arginine Polymorph Ia—Isopropanol Antisolvent

1) 20 g of tazobactam acid and 1 equivalent of L-arginine was placed into a 1 liter reactor.

2) 5 volumes (ca. 100 ml) of water (relative to tazobactam acid weight) was added to the reactor and stirred at 300 rpm (25° C.) for 10-15 minutes in order to obtain complete dissolution.

3) A further 0.75 vol water was then added to account for washing in sterile filtration step.

4) 6 volumes (120 ml) of ispopropanol was added over ca. 15 minutes in order to initiate precipitation.

5) The suspension was then aged for 15 minutes before cooling down to 10° C. over 1.5 hours at a rate of 10° C./hour.

6) After reaching 10° C., isopropanol addition was started at a rate of 185 ml/hour, adding a further 16 volumes (i.e., total isopropanol added throughout reaction was 24 volumes).

7) The reaction was then allowed to age for 1 hour.

8) The suspension was filtered and washed with 5 volumes of cold isopropanol (ca. 4° C.).

9) The resulting solid was then dried at ambient under vacuum and nitrogen bleed for 24 hours with regular mixing of the solids to yield tazobactam arginine crystalline polymorph Ia.

Example 6 Hygroscopicity Experiments

Analysis by DVS was performed on a VTI SGA-100 water vapor sorption analyser using approximately 18-23 mg of each sample. Each sample was pre-dried at 60° C. for a maximum of 1 hour and then analyzed at 25° C. in 10% relative humidity (RH) steps from 5-95% (adsorption) and then 90-10% RH (desorption). The sample was equilibrated to each humidity level for a maximum of 3 hours or until constant weight was attained. Equilibrium criteria were set at less than 0.0050% weight change within 5 minutes.

(6a) Tazobactam arginine amorphous exhibited approximately 10.7% weight increase at 95% relative humidity (RH). See FIG. 8.

(6b) Tazobactam arginine polymorph la exhibited approximately 0.2% weight increase at 95% RH. See FIG. 9.

(6c) Amorphous tazobactam sodium exhibited approximately 80% weight increase at 95% RH. See FIG. 10.

Example 7 Solvent Solubility Screen

The solvent solubility screen was carried out using a solvent addition technique. For each of 25 solvent systems, the following procedure was used: (1) about 20 mg of crystalline tazobactam arginine was placed into a vial; (2) solvent was added to the vial in 5 volume aliquots (100 μL) until complete dissolution, or 100 volumes, had been added; (3) between additions, the sample was heated to about 50° C. in order to determine the approximate solubility at an elevated temperature; (4) if 100 volumes was reached and complete dissolution was not observed, solubility was calculated to be below that point. Results of the solvent solubility screen are shown in Table 3.

Instrumentation and Methods

I. X-Ray Powder Diffraction (XRPD) experiments were performed using a Bruker D8 Advance X-ray powder diffractometer utilizing a zero return silicon plate, a step size of 0.01°, a step time of 0.3 sec/step, Cu/Kα radiation, tube power of 40 kV/40 mA, a nickel filter, and a LynxEye high speed detector. A suitable amount of sample was placed directly on the sample holder, pressed flat to smooth, and analyzed from 3°-40° 2θ using Bragg-Brentano optics. Analysis was started immediately following sample preparation. II. Differential Scanning calorimetry (DSC) experiments were performed on a TA Instruments Q100 instrument. A temperature range of 40° C. to 300° C. with a ramp rate of 10° C./minute was utilized. Approximately 1.0 mg of sample was weighed into a tared aluminum sample pan and sealed hermetically. A small hole was pushed into the cover of the sample pan to allow for pressure release. III. Thermo Gravimetric Analysis (TGA) experiments were performed on a TA Instruments 5000 instrument from 20 to 300° C. with a heating rate of 10° C./minute for all samples. Tables

TABLE 1 XRPD Scanning Data of Tazobactam Arginine Polymorph Ia (Figure 1) Chord Mid. D (Obs. Max) Intensity % Max Int. Intensity I. Breadth 2-Theta ° Angstrom % Cps Count 2-Theta ° 4.818 18.27951 33.5 130 7043 0.166 8.978 9.83463 100.0 364 21035 0.174 9.916 8.90757 8.7 32.3 1832 0.168 11.301 7.81865 27.8 104 5844 0.167 14.521 6.09321 20.2 75.5 4251 0.108 14.902 5.93864 27.8 102 5850 0.162 15.93 5.56039 1.9 7.2 394 0.148 16.947 5.23254 1.2 4.96 253 0.169 17.581 5.04332 6.8 24.8 1429 0.182 18.046 4.91261 48.7 184 10242 0.189 18.863 4.70152 2.6 9.41 545 0.159 19.418 4.5672 31.6 115 6637 0.166 19.943 4.44853 9.3 33.8 1966 0.181 21.31 4.1658 41.4 151 8714 0.192 22.797 3.89704 9.1 33.2 1921 0.201 23.587 3.76939 14.7 53.1 3082 0.171 24.345 3.65381 19.6 71.2 4116 0.208 25.169 3.53603 2.3 8.44 479 0.185 25.895 3.43955 5.4 19.7 1129 0.152 26.221 3.39654 5.0 15.6 1061 0.146 26.689 3.33736 11.1 40 2329 0.192 27.249 3.27088 5.0 19.1 1052 0.25 28.09 3.17445 5.6 20.2 1184 0.269 28.886 3.08881 3.2 11.4 666 0.219 30.129 2.96435 4.2 15.6 884 0.184 30.585 2.92187 1.8 6.17 369 0.313 31.413 2.84617 5.6 20.1 1174 0.212 32.162 2.78029 2.8 9.87 583 0.285 33.878 2.64293 1.1 2.36 236 0.109 34.419 2.60386 3.2 11.5 676 0.239 35.529 2.52408 6.0 21.9 1254 0.344 36.598 2.45267 3.0 11 621 0.269 37.924 2.37119 1.8 6.41 371 0.276 38.818 2.31643 1.4 2.74 295 0.172 39.398 2.28753 1.1 3.56 236 0.196

TABLE 2 Beta-lactam compounds No. IUPAC Name CAS No. 1 (2S,5R,6R)-6-[(R)-2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido)-2- 61477-96-1 phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane- 2-carboxylic acid 2 (2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1- 61-33-6 zabicyclo[3.2.0]heptane-2-carboxylic acid 3 (5R,6S)-6-[(1R)-1-hydroxyethyl]-3-({2-[(iminomethyl)amino]ethyl}thio)- 74431-23-5 7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4 (5R,6S)-6-((R)-1-hydroxyethyl)-7-oxo-3-((R)-tetrahydrofuran-2-yl)-4- 106560-14-9 thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid 5 (2S,5R,6R)-6-{[3-(2-chlorophenyl)-5-methyl-oxazole-4-carbonyl]amino}- 61-72-3 3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 6 (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-carboxypropan-2- 72558-82-8 yloxyimino)acetamido)-8-oxo-3-(pyridinium-1-ylmethyl)-5-thia-1-aza- bicyclo[4.2.0]oct-2-ene-2-carboxylate 7 (6R,7R,Z)-3-(acetoxymethyl)-7-(2-(2-aminothiazol-4-yl)-2- 63527-52-6 (methoxyimino)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid 8 (6R,7R)-7-[(2Z)-2-ethoxyimino-2-[5-(phosphonoamino)-1,2,4-thiadiazol- 400827-46-5 3-yl]acetyl]amino]-3-[4-(1-methylpyridin-1-ium-4-yl)-1,3-thiazol-2- yl]sulfanyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 9 (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)- 88040-23-7 3-((l-methylpyrrolidinium-1-yl)methyl)-8-oxo-5-thia-1-aza- bicyclo[4.2.0]oct-2-ene-2-carboxylate 10 (6R,7R)-3-{[(aminocarbonyl)oxy]methyl}-7-{[(2Z)-2-(2-furyl)-2- 55268-75-2 (methoxyimino) acetyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene- 2-carboxylic acid 11 (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2- 73384-59-5 (methoxyimino)acetyl]amino}-3-{[(2-methyl-5,6-dioxo-1,2,5,6- tetrahydro-1,2,4-triazin-3-yl)thio]methyl}-8-oxo-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 12 (2S,5R,6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl]amino}-3,3- 26787-78-0 dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 13 3-[5-(dimethylcarbamoyl) pyrrolidin-2-yl]sulfanyl-6-(1-hydroxyethyl)-4- 119478-56-7 methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid

TABLE 3 Solvent solubility screen results Solubility @ Solubility @ Solvent 22° C. (mg/ml) 50° C. (mg/ml) Acetic acid >100 mg/ml  >200 mg/ml  Acetone <10 mg/ml <10 mg/ml Acetonitrile <10 mg/ml <10 mg/ml 1-butanol <10 mg/ml <10 mg/ml Cyclohexane <10 mg/ml <10 mg/ml Dichloromethane <10 mg/ml <10 mg/ml Diisopropyl ether <10 mg/ml <10 mg/ml Dimethylformamide <10 mg/ml <10 mg/ml Dimethylsulfoxide <10 mg/ml ca.10 mg/ml  1,4-Dioxane <10 mg/ml <10 mg/ml Ethanol <10 mg/ml <10 mg/ml Ethyl acetate <10 mg/ml <10 mg/ml Heptane <10 mg/ml <10 mg/ml Methanol <10 mg/ml <10 mg/ml Isopropyl acetate <10 mg/ml <10 mg/ml Methyl acetate <10 mg/ml <10 mg/ml Methylethyl ketone <10 mg/ml <10 mg/ml Methyl isobutyl ketone <10 mg/ml <10 mg/ml N-Methyl-2-pyrrolidone <10 mg/ml <10 mg/ml Nitromethane <10 mg/ml <10 mg/ml 2-Propanol <10 mg/ml <10 mg/ml tert-Butylmethyl ether <10 mg/ml <10 mg/ml Tetrahydrofuran <10 mg/ml <10 mg/ml Toluene <10 mg/ml <10 mg/ml Water >100 mg/ml  >200 mg/ml  

The invention claimed is:
 1. A composition comprising crystalline tazobactam arginine characterized by an X-ray powder diffraction pattern having peaks expressed in degrees 2-Theta±0.2° at angles of 4.8°, 8.9°, 11.3°, 14.9°, 18.0°, 19.4°, 21.3°, 22.8° and 24.3°.
 2. The composition of claim 1, further characterized by a differential scanning calorimetry thermogram having a characteristic peak expressed in units of ° C. at a temperature in the range of about 209.2 to about 211.9.
 3. The composition of claim 1, further characterized by a thermogravimetry curve with an onset temperature of about 201.9° C. 